WO2011094645A1 - Méthodes de diagnostic de maladies neurodégénératives associées à tau - Google Patents

Méthodes de diagnostic de maladies neurodégénératives associées à tau Download PDF

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WO2011094645A1
WO2011094645A1 PCT/US2011/023065 US2011023065W WO2011094645A1 WO 2011094645 A1 WO2011094645 A1 WO 2011094645A1 US 2011023065 W US2011023065 W US 2011023065W WO 2011094645 A1 WO2011094645 A1 WO 2011094645A1
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tau
protein
biological sample
disease
subject
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Garth F. Hall
Wonhee Kim
Sangmook Lee
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University Of Massachusetts Lowell
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4709—Amyloid plaque core protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2828—Prion diseases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2835—Movement disorders, e.g. Parkinson, Huntington, Tourette

Definitions

  • Neurodegenerative diseases are a major public health issue. For example, an estimated 4 million Americans currently have Alzheimer's disease (AD) and by the year 2030 it is predicted that approximately 1 in every 80 persons in the U.S. will suffer from the disease.
  • AD Alzheimer's disease
  • MCI mild cognitive impairment
  • Clinicopathological studies suggest that even MCI only becomes evident a number of years after the first pathological hallmarks of AD appear (Bennett et al. Neurology 2005 vol 5 pp 834-841). Accordingly, one of the major challenges in treating neurodegenerative diseases such as AD will be to diagnose individuals prospectively, i.e. prior to the emergence of clinical symptoms, in order to begin treatment before irreversible damage has set in.
  • This invention provides methods and kits for the detection of tau- associated neurodegenerative diseases, such as Alzheimer' s disease, prior to the onset of clinical symptoms.
  • the method generally involves determining the amount of one or more tau protein isoforms in a biological sample relative to a suitable control (e.g., secreted tau), where an altered amount of the tau isoform(s) relative to the control identifies the presence of a tau-associated neurodegenerative disease.
  • a suitable control e.g., secreted tau
  • Such methods can also be applied more generally for the detection of tau abnormalities in any biological sample.
  • the invention provides methods important in the diagnosis, for example, in the early diagnosis of tau associated neurodegenerative diseases.
  • the invention provides novel biomarkers for identifying patients at risk for developing tau associated neurodegenerative disorders and particularly features detecting secreted tau as a biomarker for said diseases.
  • the invention contemplates any means of detecting secreted tau as a biomarker.
  • the invention features methods of detecting particular tau isoforms in, for example, CSF and/or methods of identifying tau as a component of the secretory apparatus of neuronal cells.
  • the invention provides methods for identifying a subject having, or at risk of developing, a tau-associated neurodegenerative disease comprising:
  • the methods of the invention can be used to diagnose a tau-associated neurodegenerative disease in any animal subject.
  • the subject is a mammal, e.g., a human.
  • the subject has no clinically discernible cognitive impairment or is pre- symptomatic (i.e., displays no symptoms of neurodegenerative disease).
  • the subject has no clinically discernable symptoms of a tau-associated neurodegenerative disease, for example, Alzheimer' s disease.
  • the subject has mild cognitive impairment (MCI) but has no clinically discernable symptoms of a tau-associated neurodegenerative disease, for example, Alzheimer' s disease.
  • the subject has mild cognitive impairment, known in the art as the stage between normal forgetfulness and dementia.
  • Any tau-associated neurodegenerative disease can be diagnosed using the methods of the invention, including, without limitation Alzheimer' s Disease, corticobasal degeneration, Pick' s Disease, progressive supernuclear palsy,
  • Creutzfeld - Jacob Disease CJD
  • Creutzfeld -Jacob Disease variant Creutzfeld -Jacob Disease
  • new variant Creutzfeld - Jacob Disease new variant Creutzfeld - Jacob Disease
  • the invention provides methods for identifying a tau abnormality in a biological sample (e.g., as a biomarker of a tau associated disorder or disease), the methods comprising:
  • the invention provides methods for identifying tau as a component of secretory vesicles of neuronal cells (e.g., as a biomarker of tau associated disorders or diseases). Such methods comprise obtaining a population of neuronal cells; determining the amount of secreted vesicular tau, for example, tau 2+ relative to a suitable control, wherein an increased amount of tau as a secreted component of said neuronal cells identifies a tau abnormality.
  • the present invention also contemplates detecting secreted tau, for example, in CSF, followed by confirmatory screening featuring detection of secreted vesicular tau.
  • any biological sample containing a tau abnormality can be assayed using the methods of the invention.
  • the biological sample is an extracellular fluid (i.e., secreted tau is detected).
  • extracellular fluids include, without limitation, cerebrospinal fluid, blood, serum and plasma.
  • the biological sample is a cell sample or media thereof or cell culture supernatant.
  • Suitable cell samples, cell media samples or cell culture supernatant include, for example, neuronal cell samples or brain cell samples.
  • the biological sample is a tissue sample or tissue lysate sample.
  • tissue samples or lysate samples include, for example brain tissue samples, tissue samples from the spinal cord or any tissue samples from the nervous system.
  • the cell, cell media sample or cell culture supernatant or the tissue or tissue lysate sample has been fractioned to separate microvesicles, for example, exosomes.
  • the instant invention features a two step method, wherein secreted tau (e.g., tau 2-, tau 2+ or a ratio of tau 2+/tau 2-) is detected in a sample (e.g., an extracellular fluid sample, e.g., CSF), the method further comprising detecting secreted tau (e.g., tau 2-, tau 2+ or a ratio of tau 2+/tau 2-) as a secretory component in a cell sample, e.g., a neuronal cell sample, for example, detection in a microsomal cell fraction, or in exosomes.
  • secreted tau e.g., tau 2-, tau 2+ or a ratio of tau 2+/tau 2-
  • a sample e.g., an extracellular fluid sample, e.g., CSF
  • secreted tau e.g., tau 2-, tau 2+ or a ratio of tau 2+/tau 2-
  • a cell sample e.g., a neuronal cell
  • the methods of the invention include assaying the biological sample for tau secretion as a biomarker for tau associated disorders or diseases.
  • tau secretion is assayed by detecting the tau isoforms in microvesicles. In certain embodiments, tau secretion is assayed by detecting tau isoforms in exosomes.
  • tau secretion is assayed by determining whether tau is present in the media that hosts a cell sample or a tissue sample.
  • the methods of the invention can employ any art recognized means for the identification of tau 2+ protein, tau 2- protein or the tau 2+/tau 2- ratio.
  • the amount of tau 2+ protein, tau 2- protein or the tau 2+/tau 2- ratio is determined by Western Blotting, ELISA, dot-blotting, high performance liquid chromatography (HPLC) and/or mass spectrometry.
  • tau 2+ protein is detected using an antibody that specifically binds to the amino acid sequence set forth in SEQ ID No.7.
  • tau 2+ protein is detected using an antibody that specifically binds to the amino acid sequence set forth in SEQ ID No.8.
  • tau 2- protein is detected using an antibody that specifically binds to the Exon 1/Exon 4 junction region of human tau. In a particular embodiment, tau 2- protein is detected using an antibody that specifically binds to the Exon 1/Exon 3 junction region of human tau.
  • the methods of the invention include assaying for tau secretion using art recognized methods, without limitation include, Western Blotting, ELISA, dot-blotting, high performance liquid chromatography (HPLC) and/or mass spectrometry.
  • the secreted tau resembles CSF tau species (e.g., cleavage fragments) associated with Alzheimer's disease.
  • tau secretion may be assayed by separating the cell media from the cells of the biological sample, assaying the cell media for the presence of tau, and comparing tau in the cell media sample to CSF tau species.
  • kits for identifying a tau abnormality in a biological sample comprising one or more means for determining the amount of tau 2+ protein, tau 2- protein or the tau 2+/tau 2- ratio, and instructions for use of the kit to identify a tau abnormality in the sample.
  • the kit includes one or more antibodies (e.g., monoclonal antibodies, polyclonal antibodies, labeled and/or unlabeled) that specifically bind to tau 2+ protein and/or tau 2- protein.
  • the kit includes an antibody that specifically binds to the amino acid sequence set forth in SEQ ID No.7.
  • the kit includes an antibody that specifically binds to the amino acid sequence set forth in SEQ ID No.8.
  • the kit includes an antibody that specifically binds to the Exonl /Exon 4 junction region of human tau. In a particular embodiment, the kit includes an antibody that specifically binds to the Exonl /Exon 3 junction region of human tau.
  • the kit further comprises a means for obtaining the biological sample.
  • the kit further comprises a suitable control sample.
  • Figure 2 depicts an amino acid sequence alignment of the N-terminal region of all six human isoforms of tau.
  • FIG. 3 schematically depicts that tau binding to microtubules is normally controlled at specific sites on tau.
  • Phosphorylation in the microtubule binding region (MTBR) (Rl, R2, R3, R4) weakens tau binding to microtubules and favors tau aggregation.
  • Phosphorylation of serine and threonine residues in the regions flanking the MTBRs reduce tau binding to microtubules and is associated with tau aggregation and toxicity.
  • Figure 4 depicts the effects of tau alterations on cytotoxicity in
  • Figure 5 schematically depicts that tau-induced degeneration is time and dose dependent and is accelerated by "tauopathy” mutations.
  • A. graphically depicts that the ability to stage tau induced degeneration in ABCs makes it possible to show that this degeneration is progressive.
  • B. graphically depicts that the rate of progression is increased with high levels of tau expressed (defined in relation to endogenous tubulin levels).
  • C. schematically depicts that the presence of tauopathy mutations (G272V, P301L, V337M, R406W) increases the proportion of high stage cells seen at both early (10-20 days of expression) and late (30+ days of expression) times after plasmid injection relative to that seen with the expression of the parent WT isoform.
  • Figure 6 depicts Western blots of cell lysates and culture media from cells expressing: A) endogenous tau; B) endogenous tubulin; C) exogenous 255 amino acid N-terminal fragments of tau in which, lacking or containing exons 2 and 3; and D) exogenous full length tau lacking or containing exons 2, or exons 2 and 3.
  • Figure 7 depicts that tau secretion from mammalian neuroblastoma (NB2A) cells also requires the tau N-terminal domain.
  • An N-terminal monoclonal antibody (tau 12, residues 9-18) recognizes secreted 1-255 and full length tau in concentrated culture medium conditioned by tau-expressing NB2A cells (asterisk).
  • Figure 8 depicts that tau secretion is inhibited by the presence of an N-terminal insert (exon 2) in MCI and NB2A cells.
  • FIG. 9 depicts that the presence of tau microtubule binding regions (MTBR) affects whether tau secretion occurs focally or in a diffuse pattern .
  • the immunoblot illustrates that secreted tau in culture occurs as two groups of bands that correspond to whether the microtubule binding region (MTBR) is present (top band - focal) or absent (bottom bands - diffuse).
  • FIG 10 A. schematically depicts the tau protein.
  • B. depicts that both diffuse and focal pathways of tau secretion from ABCs require the tau N terminal domain.
  • C depicts that overexpression of full length tau isoforms resits in focal and diffuse tau secretion.
  • D. depicts that deletion of the C terminal (MTBR) half of tau causes profuse secretion and abolishes "focal" deposits.
  • E. depicts that deletion of the N-terminal half of tau blocks secretion of tau.
  • FIG 11 depicts that tau secretion from lamprey ABCs is also inhibited by the presence of the N-terminal insert E2 in ABCs;
  • Scale Bars 100 microns.
  • Figure 12 depicts the use of monoclonal antibodies specific for E2- (secreted) and E2+ (retained) tau.
  • Monoclonal antibody 9A1 binds an epitope in the junction between exon 1 and exon 4, and is used to identify E2- (secreted) tau
  • monoclonal antibody DC39E2 binds exon 2, and is used to identify E2+ (retained) tau.
  • Figure 13 depicts the expected results of a study comparing the sensitivity of the methods of the invention with conventional CSF assays.
  • a and B show the experimental design and expected result of the proposed comparison between "total" CSF tau (All or T12+ tau), tau fragments containing the 2/3 insert (tau 2+ protein, or DC39N1+ tau) and tau phosphorylated at the AT 180 or AT270 sites (P), which currently represent the most widely used measure of "phosphorylated tau.”
  • C and D illustrate the use of an
  • Exon l/Exon4 junction region specific antibody (9A1) to directly identify tau protein fragments lacking the exon 2/3 insert (tau 2- protein or "secreted tau”) and the expected tau 2+/tau 2- ratios.
  • FIG 14 A. schematically illustrates that secreted tau is an early maker of tau-associated neurodegenerative disease, for example, Alzheimer's disease. A significant amount of secreted tau was observed in the CSF from pre-symptomatic subjects. A strong amount of secreted tau was observed in the CSF from subjects with mild cognitive impairment (MCI); B. schematically depicts the development of Alzheimer's disease as the illness proceeds from 1. The presymptomatic stage, 2. The preclinical stage, 3. Mild cognitive impairment (MCI) and finally to 4. Alzheimer's disease (AD).
  • MCI mild cognitive impairment
  • FIG. 15 depicts that secreted tau resembles cerebrospinal fluid (CSF) tau species (cleavage fragments) associated with Alzheimer's disease.
  • CSF tau cerebrospinal fluid
  • A. schematically depicts that CSF tau in AD is cleaved by disease activated enzymes (caspases and calpains) that degrade proteins at specific sites.
  • B. depicts that secreted tau occurs primarily in two fragment sizes that resemble the caspase and calpain fragments seen in AD.
  • Figure 16 depicts that tau associated with exosomes resembles CSF tau from early AD patients.
  • B. depicts secreted "large fragment” tau that is phosphorylated at the AT270 site and is selectively enriched in exosomes.
  • Figure 17 depicts the purification and characteristics of microvesicle and exosomal secreted tau.
  • A schematically depicts the purification of microvesicle secreted tau and the purification of exosomal secreted tau from media and CSF. The biological sample was subjected to several hard spins to remove large organelles from the sample. The sample was then spun in sucrose to fractionate the sample so that the fraction containing exosomes could be separated from the fraction containing other membranes, including micro vesicles.
  • B depicts that secreted tau is enriched in exosome fractions of conditioned media samples from MIC cultures induced to overexpress E2- tau as determined by mass spectrometric analysis.
  • exosomes for tau varies with tau phosphorylation state, with some phosphoepitopes (AT100 and PHFl) more prominent in secreted tau than in lysate exosome fractions and others (AT270, ATI 80) less so.
  • This figures also depicts that all Alzheimer's disease associated phosphorylation site are found in exosomal tau.
  • Figure 18 depicts that secreted tau is associated with vesicular elements positive for exosomal markers and AD-associated proteins.
  • A. depicts that tau fractions contain 80 nm vesicles positive for the exosome marker Alix; Immunoelectron microscopy (IEM) of an MIC exosome fraction showing colloidal gold decoration of the widely accepted exosome marker protein alix on 100 nm vesicles (arrows)
  • IEM Immunoelectron microscopy
  • B depicts that the proteins that co-purify with tau in exosomes are "classically" found in exosomes and/or associated with tau misprocessing in AD; tau was co-enriched with 4 classes of proteins in MIC exosomes.
  • proteins are typically exosome-associated and included some (e.g. annexin 7, alix) that are considered exosome "markers").
  • Other co-enriched proteins included known tau binding proteins (fyn kinase, HSC70 associated protein) and AD associated proteins such as APP.
  • This invention provides methods and kits for the detection of tau abnormalities in a biological sample.
  • the invention is based on the surprising finding that tau secretion can serve as a biomarker for predicting or early-stage diagnosing of tau associated disorders.
  • the invention is based, in part, on the discovery that human tau isoforms lacking the amino acids encoded by Exon 2 (herein tau 2- proteins) are secreted into the extracellular space in the brain, in a similar pattern to that seen in tau- associated neurodegenerative diseases, such as Alzheimer's disease.
  • the methods of the invention generally involve determining the amount of tau 2+ protein or tau 2- protein, or the tau 2+/tau 2- ratio in a biological sample relative to a suitable control, where a decreased amount of tau 2+ protein, an increased amount of tau 2- protein, or a decreased tau 2+/tau 2- ratio relative to the control identifies the presence of a tau abnormality.
  • Such methods and kits are particularly useful for the early detection of tau-associated neurodegenerative diseases, such as Alzheimer's disease, prior to the onset of clinical symptoms.
  • the invention is further based, at least in part, on the surprising discovery that tau isoforms, in particular, the tau 2- isoform is extracellularly secreted, for example, into the CSF by classical secretory mechanisms.
  • tau is detectable in microvesicles, or exosomes, as a step leading to secretion into, for example, CSF.
  • the invention is further based on the surprising discovery that tau secretion is a disease associated event that is a better and more useful indicator of future tau-associated neurodegenerative disease, for example, Alzheimer's disease, development than current markers of neuron death.
  • tau refers to the tau family of microtubule-associated proteins (Weingarten et al., 1975, PNAS. (72) 1858-1862), including, without limitation, the six known isoforms of human tau protein, the amino acid sequence of which have been assigned the following Genbank accession numbers: GI:82534351; GI:6754638; GI:8400711; GL8400715; GI: 178557736; and GI: 178557734.
  • tau 2+ protein refers to tau protein isoforms comprising the amino acids encoded by exon 2 of the human tau gene, or the functional equivalent of human exon 2 in other organisms.
  • tau 2- protein refers to tau protein isoforms lacking the amino acids encoded by exon 2 and exon 3 of the human tau gene, or the functional equivalent of human exon 2 and exon 3 in other organisms.
  • tau 2+/tau 2- ratio refers to the ratio of amount of tau 2+ protein to tau 2- protein in a sample.
  • tau-associated neurodegenerative disease refers to any neurodegenerative disease in which tau production, phosphorylation, aggregation or accumulation by neurons and /or glia in the CNS is abnormal. Suitable diseases include, without limitation, Alzheimer' s Disease, corticobasal degeneration, Pick' s Disease, progressive supernuclear palsy, granulovacuolar disease, frontotemporal dementia, Lewy Body disease, Creutzfeld -Jacob Disease (CJD), variant Creutzfeld -Jacob Disease, and new variant Creutzfeld -Jacob Disease.
  • CJD Creutzfeld -Jacob Disease
  • tau abnormality refers to an abnormal amount of tau 2+ protein or tau 2- protein, or an abnormal tau 2+/tau 2- ratio in a biological sample relative to a suitable control.
  • the term "amount”, with respect to tau 2+ protein or tau 2- protein refers to either (a) an absolute amount of tau 2+ protein or tau 2- protein as measured in molecules, moles or weight per unit volume or (b) a relative amount of tau 2+ protein or tau 2- protein (e.g., measured by densitometric analysis).
  • the amount of tau 2+ protein or tau 2- protein can be measured directly using standard protein assays (e.g., ELISA, mass spectrometry, and the like), or can be inferred from measuring the absolute or relative level of mRNA encoding tau 2+ protein or tau 2- protein in a sample.
  • suitable control refers to any sample or reference value useful for identifying a tau abnormality in a biological sample.
  • Suitable control samples include, without limitation, samples containing an amount of tau 2+ protein, or tau 2- protein (or possessing a tau 2+/tau 2- ratio) representative of that found in a normal subject (i.e., a subject in which tau secretion from neuronal cells is normal) and/or a subject known to have a tau- associated neurodegenerative disease (e.g., AD).
  • Suitable reference values include, without limitation, the average amount of tau 2+ protein or tau 2- protein (or the tau 2+/tau 2- ratio) obtained from a population of normal subjects and/or a population of subjects known to have a tau-associated neurodegenerative disease (e.g., AD).
  • Suitable reference values include, without limitation, the average amount of tau 2+ protein or tau 2- protein (or the tau 2+/tau 2- ratio) obtained from a population of normal subjects and/or a population of subjects known to have a tau-associated
  • AD neurodegenerative disease
  • Example 1/Exon 4 junction region refers to any epitope in the amino acid sequence encoded by the junction of exon 1 and exon 4 of human tau that is specific to tau 2- proteins.
  • Example 1/Exon 3 junction region refers to any epitope in the amino acid sequence encoded by the junction of exon 1 and exon 3 of human tau that is specific to tau 2- proteins.
  • exosome refers to a small, e.g. about 50 to about 90 nm vesicle secreted by mammalian cells. Exosomes contain a wide variety of soluble proteins including, for example, proteins whose secretion correlates with various pathological states. An exosome is created intracellularly when a segment of the cell membrane invaginates and is endocytosed. This internalized segment is ultimately broken down into smaller vesicles that are subsequently expelled from the cell.
  • Exosomes are secreted by cells under both normal and pathological conditions and, in the latter instance, present attractive candidates as detectable biomarkers.
  • the term "specifically binds to" with reference to an antibody means that the antibody exhibits appreciable affinity for a particular antigen or epitope and, generally, does not exhibit significant crossreactivity. In exemplary embodiments, the antibody exhibits no crossreactivity (e.g., an antibody that specifically binds to tau2+ protein does not crossreact with tau2- protein and vice versa).
  • "Appreciable” or preferred binding includes binding with an affinity of at least 10 6 , 10 7 , 10 8 , 10 9 M "1 , or
  • Alzheimer's Disease and related tauopathies may be used to model AD.
  • cell culture models, transgenic models and/or in situ cellular models e.g. lamprey giant neurons
  • in situ cellular models e.g. lamprey giant neurons
  • Tau proteins are microtubule-associated proteins abundant in neurons of the central nervous system (Weingarten et ah, 1975, supra). Tau proteins interact with tubulin through microtubule binding regions (MTBRs), for example, MTBRs Rl, R2, R3 and R4, to stabilize microtubules and promote tubulin assembly into microtubules.
  • MTBRs microtubule binding regions
  • Tau is phosphorylated in vivo by a host of kinases. Phosphorylation of serine and threonine residues in and flanking the MTBRs of tau weakens tau binding to
  • microtubules and is associated with tau aggregation and toxicity.
  • Hyperphosphorylation of the tau protein can result in the self-assembly of tangles of paired helical filaments and straight filaments, which are involved in the pathogenesis of Alzheimer's disease and other Tauopathies (Alonso et ah, 2001, PNAS. (98) 6923-8). Furthermore, phosphorylation of N-terminal tyrosines occurs when tau interacts with the plasma membrane and functions in the secretion and interneuronal transfer of tau. Phosphorylation of all of these sites occurs in tau- associated neurodegenerative disease, for example, Alzheimer's disease.
  • the invention is based, in part, on the discovery that human tau isoforms lacking the amino acids encoded by Exon 2 (herein tau 2- proteins) are secreted into the extracellular space in the brain, in a similar pattern to that seen in tau- associated neurodegenerative diseases, such as Alzheimer's disease.
  • tau 2- proteins human tau isoforms lacking the amino acids encoded by Exon 2
  • the presence of abnormal amounts of tau 2+ protein, tau 2- protein or an abnormal tau 2+/tau 2- ratio in a sample the amounts of tau 2+ protein or tau 2- protein are used as an indicator of tau abnormalities and for the identification of a subject having, or at risk of developing, a tau-associated neurodegenerative disease.
  • tau secretion is inhibited by the presence of an N terminal-insert (exon 2 and/or exon 3).
  • tau secretion is inhibited by the presence of the exon 2 insert.
  • Detection reagents e.g., antibodies
  • tau carrying the insert can be detected using monoclonal antibody DC39E2 which binds to exon 2.
  • tau 2+ protein also contain exon 3, and thus, detection reagents (e.g., antibodies) that specifically bind to the amino acid sequence encoded by human exon 3 can also be used to identify tau 2+ protein.
  • detection reagents e.g., antibodies
  • detection reagents that specifically bind to epitopes present in the amino acid sequence encoded by the junction of human exon 1 and exon 4 or the junction of human exon 1 and exon 3, but not present in tau 2+ proteins, can be used to specifically identify tau 2- protein.
  • tau lacking the insert can be identified using monoclonal antibody 9 A 1, which binds to epitiopes present in the junction between exon 1 and exon 4.
  • amino acid sequence encoded by human Exon 2 is as follows:
  • amino acid sequence encoded by human Exon 3 is as follows:
  • DVTAPLVDEGAPGKQAAAQPHTEIPEGTT (SEQ ID No.8).
  • tau inclusions can result in the self-assembly of tangles of paired helical filaments and straight filaments, which are involved in the pathogenesis of Alzheimer's disease and other Tauopathies (Alonso et ah, 2001, PNAS. (98) 6923-8).
  • mutations that cause familial taupathies for example, G272V, P301L and V337M, also cause tau hyperphosphorylation.
  • N-terminal tyrosines occurs when tau interacts with the plasma membrane and functions in the secretion and interneuronal transfer of tau.
  • N-terminal and C-terminal phpsphorylation sites occurs in tau-associated neurodegenerative disease, for example, Alzheimer's disease.
  • the invention provides methods for assaying the basic cellular and intracellular events that give rise to neuronal death and dementia in tau-associated neurodegenerative disease, for example, Alzheimer' s disease by analyzing the tau protein.
  • the fate and modifications of the pool of tau that is not bound to microtubules, but is associated with toxicity and degeneration is analyzed.
  • the invention provides methods for identifying a subject having, or at risk of developing, a tau-associated neurodegenerative disease.
  • Such diseases include, without limitation, Alzheimer' s Disease, corticobasal degeneration, frontotemporal lobar degeneration (also known as Pick' s Disease), progressive supernuclear palsy, granulovacuolar disease, frontotemporal dementia, Lewy Body disease, Creutzfeld - Jacob Disease (CJD), variant Creutzfeld -Jacob Disease, and new variant Creutzfeld - Jacob Disease.
  • Tau-associated neurodegenerative diseases of non-human animals including, without limitation, mad cow disease or scrapie, can also be diagnosed by the methods of the invention using the appropriate test reagents.
  • AD Alzheimer's disease
  • cognitive deficits that affect the performance of routine skills or tasks, problems with language, disorientation to time or place, poor or decreased judgment, impairments in abstract thought, loss of motor control, mood or behavior alteration, personality change, or loss of initiative.
  • the number deficits or the degree of the cognitive deficit displayed by the patient usually reflects the extent to which the disease has progressed. For example, the patient may exhibit only a mild cognitive impairment, such that the patient exhibits problems with memory (e.g. contextual memory) but is otherwise able to function well.
  • AD dementia disorders
  • ADAS-Cog Alzheimer's disease Assessment Scale-Cognitive
  • the ADAS-Cog is an 11-part test that takes 30 minutes to complete.
  • the ADAS-Cog is a preferred brief exam for the study of language and memory skills. See Rosen et al. (1984) Am J Psychiatry. 141(11): 1356-64; Ihl a/. (2000) Neuropsychobiol. 41(2): 102- 7; and Weyer et al. (1997) Int Psychogeriatr. 9(2): 123-38.
  • the Blessed Test is another quick (-10 minute) test of cognition which assesses activities of daily living and memory, concentration and orientation. See captivating et al. (1968) Br J Psychiatry 114(512):797-811.
  • the Cambridge Neuropsychological Test Automated Battery (CANTAB) is used for the assessment of cognitive deficits in humans with neurodegenerative diseases or brain damage. It consists of thirteen interrelated computerized tests of memory, attention, and executive function, and is administered via a touch sensitive screen from a personal computer. The tests are language and largely culture free, and have shown to be highly sensitive in the early detection and routine screening of Alzheimer's disease. See Swainson et al. (2001) Dement Geriatr Cogn Disord. ; 12:265-280; and Fray and Robbins (1996) Neurotoxicol rerato/.18(4):499-504. Robbins et al. (1994) Dementia 5(5):266-81.
  • the Mini Mental State Exam developed in 1975 by Folestein et al, is a brief test of mental status and cognition function. It does not measure other mental phenomena and is therefore not a substitute for a full mental status examination. It is useful in screening for dementia and its scoring system is helpful in following progress over time.
  • the Mini-Mental State Examination MMSE is widely used, with norms adjusted for age and education. It can be used to screen for cognitive impairment, to estimate the severity of cognitive impairment at a given point in time, to follow the course of cognitive changes in an individual over time, and to document an individual's response to treatment. Cognitive assessment of subjects may require formal
  • the Seven-Minute Screen is a screening tool to help identify patients who should be evaluated for Alzheimer's disease.
  • the screening tool is highly sensitive to the early signs of AD, using a series of questions to assess different types of intellectual functionality.
  • the test consists of 4 sets of questions that focus on orientation, memory, visuospatial skills and expressive language. It can distinguish between cognitive changes due to the normal aging process and cognitive deficits due to dementia. See Solomon and Pendlebury (1998) Fam Med. 30(4):265-71, Solomon et al. (1998) Arch Neurol. 55(3):349-55.
  • AD Alzheimer's disease
  • AD Alzheimer's disease
  • AD is the most common form of dementia. AD is an incurable, degenerative and terminal disease.
  • the two hallmarks of Alzheimer's Disease (AD) are the senile plaques and neurofibrillary tangles that spread through the brain as the disease develops and cause progressive dementia.
  • Neurofibrillary tangles are fibrillar intracellular aggregates composed mainly of abnormally polarized, phosphorylated and truncated tau protein.
  • SPs Senile Plaques
  • AD Alzheimer's disease
  • tau protein and beta amyloid are the central factor driving neurodegeneration in AD. They may also play a critical role in how AD lesions spread within the brain. Furthermore, during AD pathogenesis abnormalities in tau occur 'downstream" of amyloid beta.
  • the basic cellular and intercellular events that give rise to neuronal death and dementia in AD have been analyzed by studying the tau protein. It is known, for example, that a pool of tau that is not bound to microtubules is associated with toxicity and degeneration. Accordingly, studies have been performed to analyze the fate and modifications of tau.
  • the binding of tau to microtubules (MT) is normally controlled by
  • phosphorylation at specific sites on tau may play a role in the secretion and interneuronal transfer of tau.
  • phosphorylation at 9G3 occurs when tau interacts with the plasma membrane.
  • Microtubule binding regions are located near the C-terminus of the tau protein and include the following regions: Rl, R2, R3 and R4. Each of these regions (Rl-4) bind microtubules and stabilize them.
  • the instant invention provides that phosphorylation of Ser/Thr residues in the regions flanking the microtubule binding regions (MTBRs) reduce tau binding to MTs and is associated with tau aggregation and toxicity. Thus, phosphorylation in the MTBRs weakens tau-MT binding and favors tau aggregation.
  • the phosphorylation sites in and around the MTBRs include AT8, AT100, ATI 80, G272V, P301L, V37M and PHFl.
  • phosphorylation at least at one or more of these sites occurs in subjects with Alzheimer's disease ( Figures 3 and 4)
  • the methods of the invention are employed to detect the presence of abnormal amounts of tau 2+ protein or tau 2- protein, or an abnormal tau 2+/tau 2- ratio in a sample.
  • the methods of the invention are employed to detect secreted tau, for example, the tau 2- protein, in subjects who are pre- symptomatic for tau-associated neurodegenerative disease, for example, Alzheimer's disease.
  • the methods of the invention are employed to detect secreted tau, for example, the tau 2- protein, in subjects who are preclinical for tau- associated neurodegenerative disease, for example, Alzheimer's disease.
  • the methods of the invention are employed to detect secreted tau, for example, the tau 2- protein, in subjects with mild-cognitive impairment or with mild Alzheimer's disease.
  • a sample may include, without limitation, blood serum or blood plasma, CSF, urine, cell culture supernatant and other liquid samples of biological origin from an individual or a set of individuals.
  • methods of the invention are employed to detect tau in the secretory apparatus of a cell, e.g., a neuronal cell.
  • tau secretion as evidenced by detection of tau in an exosome or microsomal fraction of a cell, e.g., a neuronal cell, can be used as a biomarker for tau associated disorders or diseases.
  • Samples may have been manipulated in any way after their procurement, for example, by electrical, chemical and/or mechanical treatments.
  • the sample may be treated with reagents, solublization, or enriched for certain components, such as proteins or peptides.
  • the tau 2+ protein or tau 2- protein can be detected by any suitable method including, without limitation, immunological-based methods, optical methods, fluorescent detection, spectrometric detection, chemilumine scent detection, matrix assisted laser desorption-time-of flight (MALDI-TOF) detection, high pressure liquid chromatographic detection (HPLC), charge detection, mass detection, radio frequency detection, or light diffraction detection.
  • immunological-based methods optical methods
  • fluorescent detection spectrometric detection
  • chemilumine scent detection chemilumine scent detection
  • MALDI-TOF matrix assisted laser desorption-time-of flight
  • HPLC high pressure liquid chromatographic detection
  • charge detection mass detection
  • radio frequency detection radio frequency detection
  • light diffraction detection light diffraction detection.
  • any of a variety of known immunoassay methods can be used for detection and quantification of the tau 2+ protein or tau 2- protein including, but not limited to: immunoassay (e.g., by enzyme-linked immunosorbent assay (ELISA)) using an antibody that specifically binds to the tau 2+ protein (e.g., an antibody that specifically binds to the amino acid sequence set forth in SEQ ID No.7) or tau 2- protein (e.g., an antibody that specifically binds to the Exonl /Exon 4 junction region of human tau); and functional assays for tau 2+ protein or tau 2- protein, e.g., microtubule binding activity.
  • immunoassay e.g., by enzyme-linked immunosorbent assay (ELISA)
  • ELISA enzyme-linked immunosorbent assay
  • Immunofluorescence assays can be easily performed on CSF using a labeled antibody. It is also possible to perform such assays in blood, serum or blood plasma, urine if sufficient tau 2+ protein or tau 2- protein diffuses from human CSF to the plasma.
  • the immunocomplex (bound antibody and sample) may be further exposed to a second antibody (e.g., a reporter antibody), which is labeled and binds to the first antibody or to the biomarker.
  • a second antibody e.g., a reporter antibody
  • the secondary antibody comprises a detectable moiety, e.g., with a fluorescent marker so it can be easily visualized by any method (e.g., by eye, microscope, or machine).
  • a sandwich ELISA or modified ELISA is used.
  • such methods comprise contacting the sample with an antibody that specifically binds to the tau 2+ protein or tau 2- protein.
  • the antibody utilized may be any antibody, such as for example, monoclonal antibodies immobilized to a support. After allowing the sample time to bind with the antibody and washing of unbound sample, a labeled antibody is contacted with the sample or, in various embodiments, the capture antibody and sufficient time is allowed for the labeled antibody to specifically bind to the tau 2+ protein or tau 2- protein or the capture antibody. The bound label is detected and thus the tau 2+ protein or tau 2- protein is detected and can be quantified.
  • the tau 2+ protein or tau 2- protein is detected by mass spectrometry, or methods that employs a mass spectrometer to detect gas phase ions.
  • Mass spectrometry methods are particularly useful for use in the methods of the invention because they allow for the simultaneous detection of tau 2+ and tau 2- proteins. Hence the tau 2+/tau 2- ratio can be easily determined in a single assay.
  • mass spectrometers are time-of-flight, magnetic sector, quadrupole filter, ion trap, ion cyclotron resonance, electrostatic sector analyzer and hybrids of these.
  • the relative levels of tau 2+ protein or tau 2- protein in a sample can be determined with mass spectrometry where a standard curve can be generated using corresponding synthetic peptides without isotope labeling.
  • the tau 2+ protein or tau 2- protein in the sample can be identified and quantified when the identical synthetic peptides are isotope labeled and spiked in the sample.
  • the mass spectrometer may be a laser desorption/ionization mass spectrometer.
  • the analytes are placed on the surface of a mass spectrometry probe, a device adapted to engage a probe interface of the mass spectrometer and to present an analyte to ionizing energy for ionization and introduction into a mass spectrometer.
  • a laser desorption mass spectrometer employs laser energy, typically from an ultraviolet laser, but also from an infrared laser, to desorb analytes from a surface, to volatilize and ionize them and make them available to the ion optics of the mass spectrometer.
  • the sample obtained from an individual is contacted with an adsorbent surface for a period of time sufficient to allow tau 2+ protein or tau 2- protein present in the sample to bind to the adsorbent surface. After an incubation period, the substrate is washed to remove unbound material. Any suitable washing solutions can be used; such as an aqueous solution. The extent to which molecules remain bound can be
  • the elution characteristics of a wash solution can depend, for example, on pH, ionic strength, hydrophobicity, degree of chaotropism, detergent strength, and temperature.
  • An energy absorbing molecule is then applied to the substrate and the bound tau 2+ protein or tau 2- protein is then detected in a gas phase ion spectrometer such as a time-of-flight mass spectrometer or an ion trap mass spectrometer.
  • the tau 2+ protein or tau 2- protein is ionized by an ionization source such as a laser, the generated ions are collected by an ion optic assembly, and then a mass analyzer disperses and analyzes the passing ions.
  • the detector then translates information of the detected ions into mass-to-charge ratios. Detection of tau 2+ protein or tau 2- protein may involve the detection of the signal intensity. Thus, both the quantity and mass of the tau 2+ protein or tau 2- protein can be determined.
  • tau 2+ protein or tau 2- protein may be first captured on a chromatographic resin that binds it.
  • the resin can be derivatized with an antibody.
  • this method could be preceded by chromatographic fractionation before application to the bio-affinity resin.
  • the sample can be analyzed by MALDI, electrospray, or another ionization method for mass spectrometry.
  • MALDI electrospray
  • electrospray or another ionization method for mass spectrometry.
  • detection of tau 2+ protein or tau 2- protein can be accomplished using capture reagents that specifically bind to the tau 2+ protein or tau 2- protein (e.g., an antibody that specifically binds to the amino acid sequence set forth in SEQ ID No.7 or the Exonl /Exon 4 junction region of human tau).
  • the capture reagent may be bound (e.g., covalently or non-covalently, via hydrophobic or hydrophilic interactions, H bonding, or van der Waals etc.) to a solid phase, such as a bead, a plate, a membrane or a chip.
  • Methods of coupling biomolecules, such as antibodies or antigens, to a solid phase are well known in the art. They can employ, for example, bifunctional linking agents, or the solid phase can be derivatized with a reactive group, such as an epoxide or an imidizole, that will bind the molecule on contact.
  • biochips may be employed.
  • the surfaces of biochips may be derivatized with the capture reagents directed against the tau 2+ protein or tau 2- protein.
  • Biochips generally comprise solid substrates and have a generally planar surface, to which a capture reagent (also called an adsorbent or affinity reagent) is attached.
  • a capture reagent also called an adsorbent or affinity reagent
  • the surface of a biochip comprises a plurality of addressable locations, each of which has the capture reagent bound thereto.
  • addressable arrays can be created to capture, detect and quantify one or more biomarkers in addition to the tau 2+ protein or tau 2- protein.
  • Exosomes can be purified using any one of a variety of art-recognized methods. In general, exosomes are purified from lysed cell samples by differential centrifugation and require, for example, sucrose gradient centrifugation to achieve purity and homogeneity of the purified exosome preparations. Exemplary techniques can be found, for example, in "Isolation and Characterization of Exosomes from Cell Culture
  • the amino acid sequence of the region of tau analogous to human exon 2 or the human exon 1-4 junction region in that non-human subject can be used as readouts for tau 2+ proteins and tau 2- proteins respectively.
  • Such analogous regions can be easily identified by, for example, sequence similarity algorithms (e.g., BLAST) or manual inspection of the amino acid sequence.
  • the methods of the invention can be used either alone or in concert with any one or more art recognized assays for employed for the diagnosis of tau- associated neurodegenerative diseases.
  • tau protein co-purifies with exosomes, which are a marker of a clearly characterized, yet unconventional mode of secretion from many cell types, including neurons.
  • the proteins that co-purify with tau in exosomes are mostly membrane associated proteins that are either known exosome markers such as alix and annexin 7 or are from protein families that have been shown to be in exosomes.
  • Other proteins that are co-enriched with tau in exosomes (and which themselves are known to be exosome associated) have art-recognized links to tau processing in AD.
  • fyn kinase is known to play a role in disease-associated tau misprocessing that could plausibly account for the localization of tau to exosomes in the early stages of AD and/or other tauopathies.
  • exosomal tau purified from the media of neuronal cells in culture is somewhat dephosphorylated relative to most CSF tau, strengthening the idea that it is derived from a different source than the tau found in established AD cases, which is presumably due to release after neuron death.
  • Exosomes are present in high concentration in human CSF , along with tau and other neurodegenerative disease markers, but no one has yet attempted to associate any of these markers selectively with exosomes.
  • the findings of the instant inventors a) provide a biological mechanism that confirms the validity of tau secretion, and b) greatly increases the validity of tau secretion as a disease-associated event that can serve as a better and more useful indicator of future AD development than current art markers of neuron death (e.g., phosphorylated tau).
  • tau is exosome associated. Furthermore, there has been a widespread assumption in the art that tau is cytosolic, is not secreted and that the tau commonly found in the CSF of AD patients is exclusively due to the passive release of tau from dead neurons. However, three other proteins that drive
  • AD and other neurodegenerative diseases i.e. alpha synuclein, beta amyloid and prion protein
  • tau neurodegenerative diseases
  • the invention features the novel finding that tau isoforms can be used as biomarkers for early diagnosis of Alzheimer's as certain forms of tau are secreted in CSF.
  • the instant invention also features the novel finding that tau isoforms are secreted in exosomes.
  • AD is diagnosed using a two step process.
  • the first step comprises determining if Tau isoforms are present in the CSF, and the second step comprises purifying the exosomal particles from, e.g., neuronal cells or CSF and identifying the Tau isoforms.
  • kits for identifying a tau abnormality in a biological sample comprising: a) means for determining the amount of the tau 2+ protein or tau 2- protein, or the tau 2+/tau 2- ratio; and b) instructions for use of the kit to identify a tau abnormality in the sample.
  • kits includes one or more antibodies (e.g., monoclonal antibodies, polyclonal antibodies, labeled and/or unlabeled) that specifically bind to tau 2+ protein and/or tau 2- protein.
  • a supplied antibody specifically binds to the amino acid sequence set forth in SEQ ID No.7.
  • a supplied antibody specifically binds to the amino acid sequence set forth in SEQ ID No.8.
  • the supplied antibody specifically binds to the Exonl /Exon 4 junction region of human tau.
  • the supplied antibody specifically binds to the Exonl /Exon 3 junction region of human tau.
  • the kit may optionally provide additional components that are useful in the procedure, including, but not limited to, buffers, developing reagents, labels, reacting surfaces, control samples, and standards.
  • the kit may further provide a means for isolating the biological sample.
  • Plasmids used in this study were derived from pRc/CMVnl23c and
  • pRcCMVnl23 As described in Arai et al. 2004, Ann. Neurol. 55, 72-79. Both plasmids lack two N-terminal insert regions and contain three or four microtubule binding repeat sequences respectively.
  • GFP green fluorescence protein
  • T23 no N-terminal insert and three C-terminal repeats
  • T24 no N- terminal insert and four C-terminal repeats.
  • N-terminal and C-terminal constructs express 1-255 and 211-441 amino acids respectively of T23.
  • the GFP/T23 bicistronic construct expresses GFP from the SV40 promoter and T23 from the CMV promoter separately.
  • Transfection of N2BA cells was carried out using LipofectamineTM 2000 (Sigma Aldrich) according to the manufacturer's protocol. Serum- free medium was replaced with complete medium 24 hours after the transfection. Successfully transfected cells were localized by GFP fluorescence. Transfection rates under this condition routinely exceeded 70%. Culture medium was collected 24 hours after medium replacement and cleared by centrifugation at 10,000 x g at 4°C for 10 minutes to remove cells and cellular debris. To concentrate protein, Centricon (MiUipore, Billerica, MA) was used according to the manufacturer's protocol.
  • NB2a/dl cells were lysed in Tris-NaCl (TN) buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton-X, 10% Glycerol, 2 mM EDTA, protease inhibitor cocktail (Sigma protease inhibitors). Cell lysates were clarified by centrifugation at 10,000 x g for 10 minutes.
  • TN Tris-NaCl
  • Plasmid microinjection was performed as previously described in Lee et. al 2009. J. Alz Dis vol 16, pp 99-111, 2008. Neurobiology of Aging 30, 34-40. Briefly, an anesthetized lamprey hindbrain was exposed and the identified anterior bulbar cells were injected with the plasmid with 0.5% fast green at a final concentration of lmg/ml. A total of approximately 1100 lampreys were used for in situ microinjections, with an expression rate of between 1-2 anterior bulbar cells per lamprey injected. A total of 460 tau-expressing cells were identified for use in this study. Immunohistochemistry
  • Lamprey ABCs possess the ideal combination of accessibility, large size and stereotyped morphology for use in an in situ cellular model.
  • Normal ABC morphology features large and ellipsoidal somata, axons which are straight unbranched processes that extend caudally and ipsilaterally from the soma along the midline in the ventral tracts for most of the length of the spinal cord and dendrites, which are divided into two major domains, one extending laterally from the soma and the other medially.
  • the lateral dendritic domain consists of 1 or 2 thick profusely branched primary dendrites which extend from the lateral aspect of the soma for approximately 150 m before turning ventromedially.
  • the medial domain is smaller and more variable, comprising one or a few primary dendrites extending ventromedially from the soma for 50-100 m.
  • Dendritic degeneration occurs over several weeks following expression of exogenous tau via plasmid injection, beginning with the distal most dendrites (e.g., at about 17 days) and proceeding proximally with time (e.g., at about 28 days).
  • Figure 7 also illustrates that the N-terminal mAb tau 12 (residues 9-18) recognizes secreted 1-255 and full length tau in concentrated culture medium
  • FIG 8 clearly shows that in the presence of an N-terminal insert (E2), tau secretion is inhibited in both MIC and NB2A cells. Furthermore, this observation was found to be true for both IP and centrifuge-cencentrated media samples, cells with transient or induced tau expression and full length and N-terminal tau constructs. The data also clearly illustrate that tau without the E2 insert is secreted into the media 10-15 times more efficiently that tau comprising an E2 insert. (Figure 8b).
  • E2 N-terminal insert
  • This Example illustrates the mechanisms of tau secretion in lamprey ABCs and human neuroblastoma (MIC) cells.
  • Expression of full length human tau isoforms (T23, T24, T40) in ABCs produces 2 tau secretion patterns referred to as diffuse and focal deposits.
  • Diffuse Tau deposits are broadly distributed perisomatic deposits. Focal deposits are localized around degenerating dendrites near the point of onset of degenerations (POD). It is noteworthy that degeneration then proceeds along a distal to proximal gradient with time.
  • tau secretion correlates with AD onset in human CSF and brain samples, and will be analyzed using commercially available monoclonal antibodies specifis for E2- (secreted) and E2+ (retained) tau (Figure 12).
  • CSF samples from MCI i.e. prodromal AD
  • confirmed AD patients can be obtained from the Alzheimer's Brain Bank at Boston University Medical College (BUMCBB).
  • CSF samples from normal, cognitively intact, age matched individuals can be used as negative controls, and will be expected to contain low levels of tau 2- protein (9A1+), tau 2+ protein (DC39E2+) and total tau (Taul2).
  • CSF samples from patients with severe head trauma who are known to have suffered widespread neuronal loss typically exhibit high CSF-tau levels can be used as positive controls for neuronal death, and will be expected to show relatively high levels of 2+ tau, since tau release after TBI should be due to nonspecific leakage of tau from large numbers of ischemic, dying neurons.
  • Samples from MCI and AD patients with history of stroke, multi-infarct dementia or other conditions associated with CNS ischemia can, therefore, be excluded from the study.
  • the diagnostic efficacy of the methods of the invention as compared to conventional methods can be assessed by comparing the tau 2+/2- ratio in CSF samples with AT 180+ and AT270+ levels (the current standard measure of CSF tau
  • CSF samples should reflect the onset of degenerative changes leading to dementia but without widespread neuronal death, and thus should contain a highly significant increase in secreted tau 2- protein to the CSF (Figure 13B,**). This may be accompanied by some neuronal death and possibly also by some secretion of Ptau/ tau 2- protein, accounting for the observed increase in Ptau ( Figure 13B, *) that underlies the diagnostic value of current Ptau/total tau-based assays in MCI patients.
  • the tau 2+/2- ratio should be extremely sensitive to early changes associated with MCI, and the results should clearly indicate that a tau 2+/2- ratio-based diagnostic is more effective than current methodologies at diagnosing early stage tau- associated neurodegenerative disease (Figure 13).
  • FIG. 14a illustrates that the secreted tau protein may be the earliest biomarker of AD.
  • CSF cerebrospinal fluid
  • the AD-associated protein tau should be secreted to the CSF before degeneration onset in subjects with AD. This invention should allow for the identification of AD patients before symptoms occur and is suitable for widespread screening of individuals at high risk for AD.
  • this invention should be advantageous over current AD diagnostics which rely on neuron death markers in the CSF, which prevents the detection of AD before neuron loss occurs.
  • current diagnostics use "death" biomarkers (e.g.
  • phosphorylated tau can only identify AD after the onset of "mild cognitive impairment” (MCI).
  • MCI reduced cognitive impairment
  • identifying subjects with AD before irreversible changes occur may: 1. make treatments more effective, 2. identify pre-symptomatic AD patients for inclusion in current drug trials, and 3. identify "curable" AD patients for treatment in the future.
  • E2-tau an identifiable AD biomarker secreted from neurons before they degenerate will help improve diagnostics for AD. Furthermore, if secreted tau precedes "death" markers such as P-tau, E2-tau should be enriched in CSF samples from MCI/early AD patients.
  • Figure P further illustrates that secreted tau associated with exosomes resembles CSF tau from early AD patients. Furthermore, these data show that the secreted "large fragment" tau that is phosphorylated at the AT270 site is selectively enriched in exosomes (Figure 16).
  • Figure 17 schematically depicts the purification of micro vesicle secreted tau and the purification of exosomal secreted tau from media and CSF
  • proteins co-purified in media exosome fractions with tau were specifically identified based on sequence databases 50 . Most of these were membrane associated proteins with signal transduction functions, including several specific markers of exosomes (e.g. Alix - Figure 18a). Almost all of the proteins (with the notable exception of tau) are members of families that have been identified in exosome preparations from various tissues and bodily fluids (neuroblastoma, urine, blood, cerebrospinal fluid) in earlier studies. Of these, 21 proteins which were both a) consistently seen in MIC media exosome fractions in significant amounts (top quartile peak height) and b) enriched in media exosomes relative to their concentration in exosome preparations from MIC lysates.
  • this Example further illustrates that tau secretion in lamprey occurs via vesicles containing fyn, an exosomal protein associated with AD. It was observed that the tyrosine kinase fyn, which selectively phosphrorylates tau in AD, colocalizes at the vesicles that are undergoing exocytosis from ABC dendrites.

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Abstract

La présente invention concerne des méthodes et des kits permettant la détection de maladies neurodégénératives associées à tau, comme la maladie d'Alzheimer, avant le déclenchement des symptômes cliniques. La méthode consiste de façon générale à déterminer la quantité d'une ou plusieurs isoformes de la protéine tau dans un échantillon biologique par rapport à un témoin approprié, une modification de la quantité de l'isoforme ou des isoformes de tau par rapport au témoin identifiant la présence d'une maladie neurodégénérative associée à tau. De telles méthodes peuvent également être appliquées de façon plus générale à la détection d'anomalies de la protéine tau dans n'importe quel échantillon biologique.
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WO2015061634A3 (fr) * 2013-10-24 2015-10-29 Nanosomix, Inc. Biomarqueurs et procédés de diagnostic pour la maladie d'alzheimer et d'autres troubles neurodégénératifs
WO2015068075A3 (fr) * 2013-10-24 2015-10-29 Institut National De La Sante Et De La Recherche Medicale Détection de protéine tau
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